Effect of periodontal treatment on the C-reactive protein and proinflammatory cytokine levels in Japanese periodontitis patients

Background: Recent epidemiological studies have shown that individuals with periodontitis have a significantly increased risk of developing coronary heart disease. In addition to conventional risk factors, chronic infection and subsequent production of systemic inflammatory markers may be associated with this increased risk. Objectives: The aim of the present study was to determine whether the presence of chronic periodontitis and subsequent periodontal treatment could influence the serum levels of C-reactive protein (CRP), interleukin-6 and tumor necrosis factor-alpha (TNF-alpha) in a Japanese population. Methods: Sera were obtained from 24 patients with moderate to advanced periodontitis at the baseline examination and at reassessment after completion of treatment. As a control, sera were also obtained from 21 subjects without periodontitis. High-sensitivity CRP (hs-CRP) was measured using nephelometry with a latex particle-enhanced immunoassay and interleukin-6 and TNF-alpha were determined by sensitive enzyme-linked immunosorbent assay. Results: The levels of hs-CRP and interleukin-6 in the sera of this Japanese population seemed to be much lower than those reported in other populations. TNF-alpha on the other hand, demonstrated similar levels between this Japanese and other populations. Periodontal status demonstrated a significant improvement in all patients following treatment. There was a trend toward higher hs-CRP levels in patients at baseline compared with control subjects. Hs-CRP level tended to decrease with improvement of the periodontal condition following treatment and approached that of control subjects, although this decline was not statistically significant. interleukin-6 and TNF-alpha levels did not change following periodontal treatment. Furthermore, there was no difference in the serum levels of these inflammatory cytokines between patients either at baseline or at reassessment and control subjects. Conclusions: In this pilot study, we were unable to show that periodontal disease significantly affects the serum levels of systemic inflammatory markers. However, this does not necessarily mean that periodontitis does not contribute to the total burden of inflammation as there was a tendency for hs-CRP to decrease following successful periodontal treatment. Large-scale studies are clearly needed to determine the impact of periodontal disease on systemic inflammation.

Regulation of antigen processing and presentation molecules in West Nile virus-infected human skin fibroblasts

Infection of humans with the West Nile flavivirus principally occurs via tick and mosquito bites. Here, we document the expression of antigen processing and presentation molecules in West Nile virus (WNV)-infected human skin fibroblast (HFF) cells. Using a new Flavivirus-specific antibody, 4G4, we have analyzed cell surface human leukocyte antigen (HLA) expression on virus-infected cells at a single cell level. Using this approach, we show that West Nile Virus infection alters surface HLA expression on both infected HFF and neighboring uninfected HFF cells. Interestingly, increased surface HLA evident on infected HFF cultures is almost entirely due to virus-induced interferon (IFN)alpha/beta because IFNalpha/beta-neutralizing antibodies completely prevent increased surface HLA expression. In contrast, RT-PCR analysis indicates that WNV infection results in increased mRNAs for HLA-A, -B, and -C genes, and HLA-associated molecules low molecular weight polypeptide-2 (LMP-2) and transporter associated with antigen presentation-1 (TAP-1), but induction of these mRNAs is not diminished in HFF cells cultured with IFNalpha/beta-neutralizing antibodies. Taken together, these data support the idea that that both cytokine-dependent and cytokine-independent mechanisms account for WNV-induced HLA expression in human skin fibroblasts. (C) 2004 Elsevier Inc. All rights reserved.

A role for the phagosome in cytokine secretion

Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor alpha (TNF alpha) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNF alpha and expands the membrane for phagocytosis.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha(TNF alpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNF alpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNF alpha trafficking and secretion. TNF alpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNF alpha surface distribution revealed these puncta to be the initial points of TNF alpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

Systematic inflammatory responses to maximal versus submaximal lengthening contractions of the elbow flexors

We compared changes in markers of muscle damage and systemic inflammation after submaximal and maximal lengthening muscle contractions of the elbow flexors. Using a cross-over design, 10 healthy young men not involved in resistance training completed a submaximal trial (10 sets of 60 lengthening contractions at 10% maximum isometric strength, 1 min rest between sets), followed by a maximal trial (10 sets of three lengthening contractions at 100% maximum isometric strength, 3 min rest between sets). Lengthening contractions were performed on an isokinetic dynamometer. Opposite arms were used for the submaximal and maximal trials, and the trials were separated by a minimum of two weeks. Blood was sampled before, immediately after, 1 h, 3 h, and 1-4 d after each trial. Total leukocyte and neutrophil numbers, and the serum concentration of soluble tumor necrosis factor-alpha receptor 1 were elevated after both trials (P < 0.01), but there were no differences between the trials. Serum IL-6 concentration was elevated 3 h after the submaximal contractions (P < 0.01). The concentrations of serum tumor necrosis factor-alpha, IL-1 receptor antagonist, IL-10, granulocyte-colony stimulating factor and plasma C-reactive protein remained unchanged following both trials. Maximum isometric strength and range of motion decreased significantly (P < 0.001) after both trials, and were lower from 1-4 days after the maximal contractions compared to the submaximal contractions. Plasma myoglobin concentration and creatine kinase activity, muscle soreness and upper arm circumference all increased after both trials (P < 0.01), but were not significantly different between the trials. Therefore, there were no differences in markers of systemic inflammation, despite evidence of greater muscle damage following maximal versus submaximal lengthening contractions of the elbow flexors.

The Proinflammatory Environment in Potential Heart and Lung Donors: Prevalence and Impact of Donor Management and Hormonal Therapy

BACKGROUND: Brain stem death can elicit a potentially manipulable cardiotoxic proinflammatory cytokine response. We investigated the prevalence of this response, the impact of donor management with tri-iodothyronine (T3) and methylprednisolone (MP) administration, and the relationship of biomarkers to organ function and transplant suitability. METHODS: In a prospective randomized double-blinded factorially designed study of T3 and MP therapy, we measured serum levels of interleukin-1 and -6 (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein, and procalcitonin (PCT) levels in 79 potential heart or lung donors. Measurements were performed before and after 4 hr of algorithm-based donor management to optimize cardiorespiratory function and +/-hormone treatment. Donors were assigned to receive T3, MP, both drugs, or placebo. RESULTS: Initial IL-1 was elevated in 16% donors, IL-6 in 100%, TNF-alpha in 28%, CRP in 98%, and PCT in 87%. Overall biomarker concentrations did not change between initial and later measurements and neither T3 nor MP effected any change. Both PCT (P =0.02) and TNF-alpha (P =0.044) levels were higher in donor hearts with marginal hemodynamics at initial assessment. Higher PCT levels were related to worse cardiac index and right and left ventricular ejection fractions and a PCT level more than 2 ng x mL(-1) may attenuate any improvement in cardiac index gained by donor management. No differences were observed between initially marginal and nonmarginal donor lungs. A PCT level less than or equal to 2 ng x mL(-1) but not other biomarkers predicted transplant suitability following management. CONCLUSIONS: There is high prevalence of a proinflammatory environment in the organ donor that is not affected by tri-iodothyronine or MP therapy. High PCT and TNF-alpha levels are associated with donor heart dysfunction. (C) 2009 Lippincott Williams & Wilkins, Inc.

Induction of placental heme oxygenase-1 is protective against TNFα-induced cytotoxicity and promotes vessel relaxation

Background: Pregnancy is characterized by an inflammatory-like process and this may be exacerbated in preeclampsia. The heme oxygenase (HO) enzymes generate carbon monoxide (CO) that induces blood vessel relaxation and biliverdin that acts as an endogenous antioxidant. Materials and Methods: We examined the expression and localization of HO-1 and HO-2 in normal and preeclamptic placenta using reverse transcription polymerase chain reaction (RT-PCR), RNase protection assay, immunoblotting and immunohistochemistry. In addition, the effect of HO activation on tumor necrosis factor-alpha (TNF) induced placental damage and on feto-placental circulation was studied. Results: We provide the first evidence for the role of HO as an endogenous placental factor involved with cytoprotection and placental blood vessel relaxation. HO-1 was significantly higher at term, compared with first trimester placentae indicating its role in placental vascular development and regulation. HO-1 predominantly localized in the extravascular connective tissue that forms the perivascular contractile sheath around the developing blood vessels. HO-2 was localized in the capillaries, as well as the villous stroma, with weak staining of trophoblast. Induction of HO-1 caused a significant attenuation of TNF-mediated cellular damage in placental villous explants, as assessed by lactate dehydrogenase leakage (p 0.01). HO-1 protein was significantly reduced in placentae from pregnancies complicated with preeclampsia, compared with gestationally matched normal pregnancies. This suggests that the impairment of HO-1 activation may compromise the compensatory mechanism and predispose the placenta to cellular injury and subsequent maternal endothelial cell activation. Isometric contractility studies showed that hemin reduced vascular tension by 61% in U46619-preconstricted placental arteries. Hemininduced vessel relaxation and CO production was inhibited by HO inhibitor, tin protoporphyrin IX. Conclusions: Our findings establish HO-1 as an endogenous system that offers protection against cytotoxic damage in the placenta, identifies the HO-CO pathway to regulate feto-placental circulation and provides a new approach to study the disease of preeclampsia.

Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gate

Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.

A influência da perda de peso no perfil inflamatório de mulheres com síndrome dos ovários policísticos

The polycystic ovary syndrome (PCOS) is considered the most common endocrine disorder in reproductive age women, with a prevalence ranging from 15 to 20%. In addition to hormonal and reproductive changes, it is common in PCOS the presence of risk factors for developing cardiovascular disease (CVD) and diabetes mellitus, insulin resistance (IR), visceral obesity, chronic low-grade inflammation and dyslipidemia. Due to the high frequency of obesity associated with PCOS, weight loss is considered as the first-line treatment for the syndrome by improving metabolic and normalizes serum androgens, restoring reproductive function of these patients. Objectives: To evaluate the inflammatory markers and IR in women with PCOS and healthy ovulatory with different nutritional status and how these parameters are displayed after weight loss through caloric restriction in with Down syndrome. Methods: Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP) were assessed in serum samples from 40 women of childbearing age. The volunteers were divided into four groups: Group I (not eutrophic with PCOS, n = 12); Group II (not eutrophic without PCOS, n = 10), Group III (eutrophic with PCOS, n = 08) and Group IV (eutrophic without PCOS, n = 10). The categorization of groups was performed by body mass index (BMI), according to the World Health Organization (WHO) does not eutrophic, overweight and obesity (BMI> 25 kg / m²) and normal weight (BMI <24.9 kg / m²). IR was determined by HOMA-IR index. In the second phase of the study a controlled dietary intervention was performed and inflammatory parameters were evaluated in 21 overweight and obese women with PCOS, before and after weight loss. All patients received a low-calorie diet with reduction of 500 kcal / day of regular consumption with standard concentrations of macronutrients. Results: Phase 1: PCOS patients showed increased levels of CRP (p <0.01) and HOMAIR (p <0.01). When divided by BMI, both not eutrophic group with PCOS (I) as eutrophic with PCOS (III) showed increased levels of CRP (I = 2.35 ± 0,55mg / L and 2.63 ± III = 0,65mg / L; p <0.01) and HOMA-IR (I = 2.16 ± 2.54 and III = 1.07 ± 0.55; p <0.01). There were no differences in TNF-α and IL-6 between groups. Step 2: After the weight loss of 5% of the initial weight was reduced in all of the components of serum assessed inflammatory profile, PCR (154.75 ± 19:33) vs (78.06 ± 8.9) TNF α (10.89 ± 5.09) vs (6:39 ± 1:41) and IL6 (154.75 ± 19:33) vs (78.06 ± 08.09) (p <0:00) in association with improvement some hormonal parameters evaluated. Conclusion: PCOS contributed to the development of chronic inflammation and changes in glucose metabolism by increasing CRP, insulin and HOMA-IR, independent of nutritional status. The weight loss, caloric restriction has improved the inflammatory condition and hormonal status of the evaluated patients.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.

Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.

Viral Delivery of the Fat-1 Gene to Treat Post-Traumatic Arthritis with Diet-Induced Obesity

Post-traumatic arthritis (PTA) is arthritis that develops following joint injury, including meniscus and ligament tears. Current treatments for PTA range from over-the-counter medication to knee replacement; however, in the presence of obesity, the levels of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α,) are more elevated than in non-obese individuals. The role of fatty acids, obesity, and PTA has been examined, with omega-3 fatty acids showing promise as an anti-inflammatory after injury due to its ability to suppress IL-1 and TNF-α. Due to the difficulty in switching patients’ diets, an alternative solution to increasing omega-3 levels needs to be developed. The Fat-1 enzyme, an omega-3 desaturase that has the ability to convert omega-6 to omega-3 fatty acids, may be a good target for increasing the omega-3 levels in the body.

In the first study, we examined whether Fat-1 transgenic mice on a high-fat diet would exhibit lower levels of PTA degeneration following the destabilization of the medial meniscus (DMM). Both male and female Fat-1 and wild-type (WT) littermates were put on either a control diet (10% fat) or an omega-6 rich high-fat diet (60% fat) and underwent DMM surgery. Arthritic changes were examined 12 weeks post-surgery. Fat-1 mice on both the control and high-fat diet showed protection from PTA-related degeneration, while WT mice showed severe arthritic changes. These findings suggest that the omega-6/omega-3 ratio plays an important role in reducing PTA following injury, and demonstrates the potential therapeutic benefit of the Fat-1 enzyme in preventing PTA in both normal and obese patients following acute injury.

Following this, we needed to establish a translatable delivery mechanism for getting the Fat-1 enzyme, which is not present in mammalian cells, into patients. In the second study, we examined whether anti-inflammatory gene delivery of the Fat-1 enzyme would prevent PTA following DMM surgery. In vitro testing of both lentivirus (LV) and adeno-associated virus (AAV) was completed to confirm functionality and conformation of the Fat-1 enzyme after transduction. Male WT mice were placed on an omega-6 rich high-fat diet (60% fat) and underwent DMM surgery; either local or systemic AAV injections of the Fat-1 enzyme or Luciferase, a vector control, were given immediately following surgery. 12 weeks post-surgery, arthritic changes were assessed. The systemic administration of the Fat-1 enzyme showed protection from synovial inflammation and osteophyte formation, while administration of Luciferase did not confer protection. These findings suggest the utility of gene therapy to deliver the Fat-1 enzyme, which has potential as a therapeutic for injured obese patients for the prevention of PTA.

Exaggerated inflammation and monocytosis associate with diastolic dysfunction in heart failure with preserved ejection fraction: evidence of M2 macrophage activation in disease pathogenesis

BACKGROUND: Heart failure with preserved ejection fraction (HFPEF) is a major health problem associated with myocardial leukocyte infiltration, inflammation, and fibrosis. Monocyte and macrophage subsets play a role in HFPEF but have not been studied. We analyzed peripheral blood monocyte phenotype and plasma markers of monocyte activation in patients with HFPEF, asymptomatic LV diastolic dysfunction (aLVDD), and asymptomatic hypertension (aHTN).

METHODS AND RESULTS: Peripheral blood was collected from 23 aHTN, 30 aLVDD, and 30 HFPEF patients. Peripheral cytokines of classic/pro-inflammatory (tumor necrosis factor alpha, interleukin (IL) 12, IL-6, monocyte chemoattractant protein 1, C-X-C motif chemokine 10) and alternative/anti-inflammatory monocytes (chemokine-C-C motif ligand (CCL) 17, CCL-18, soluble CD163) were increased in aLVDD and HFPEF. Peripheral blood mononuclear cells and monocytes were purified and surface-stained for CD14, CD16, CD163, and CD206. Peripheral monocyte percentage was increased in aLVDD and HFPEF and correlated with echocardiographic LVDD indices. Classic/pro-inflammatory monocyte numbers were increased in aLVDD and HFPEF, and alternative/anti-inflammatory monocyte numbers were increased in HFPEF. CD163 M2-macrophage receptor was reduced in HFPEF. Culture of healthy donor monocytes (n = 3) with HFPEF patient-derived sera (n = 6) promoted M2 macrophage features as evidenced by altered morphology and genes (CD206, IL-10).

CONCLUSIONS: Increased peripheral inflammation, monocytosis, and monocyte differentiation to anti-inflammatory/profibrotic M2 macrophages likely associate with HFPEF and its precedent asymptomatic LVDD phase.